separable fragments and membrane tethering of arabidopsis ... · separable fragments and membrane...
TRANSCRIPT
Separable Fragments and Membrane Tethering of ArabidopsisRIN4 Regulate Its Suppression of PAMP-Triggered Immunity W
Ahmed J. Afzal,a,1 Luis da Cunha,a,1,2 and David Mackey,a,b,3
a Department of Horticulture and Crop Science, Ohio State University, Columbus, Ohio 43210b Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210
RPM1-interacting protein 4 (RIN4) is a multifunctional Arabidopsis thaliana protein that regulates plant immune responses
to pathogen-associated molecular patterns (PAMPs) and bacterial type III effector proteins (T3Es). RIN4, which is targeted
by multiple defense-suppressing T3Es, provides a mechanistic link between PAMP-triggered immunity (PTI) and effector-
triggered immunity and effector suppression of plant defense. Here we report on a structure–function analysis of RIN4-
mediated suppression of PTI. Separable fragments of RIN4, including those produced when the T3E AvrRpt2 cleaves RIN4
and each containing a plant-specific nitrate-induced (NOI) domain, suppress PTI. The N-terminal and C-terminal NOIs each
contribute to PTI suppression and are evolutionarily conserved. Native RIN4 is anchored to the plasma membrane by
C-terminal acylation. Nonmembrane-tethered derivatives of RIN4 activate a cell death response in wild-type Arabidopsis
and are hyperactive PTI suppressors in a mutant background that lacks the cell death response. Our results indicate that
RIN4 is a multifunctional suppressor of PTI and that a virulence function of AvrRpt2 may include cleaving RIN4 into active
defense-suppressing fragments.
INTRODUCTION
Plants rely on a layered defense system to fend off pathogen
attack. Among the active responses are those emanating from two
branches of the plants innate immune system: pathogen-associ-
ated molecular pattern (PAMP)-triggered immunity (PTI) and
effector-triggered immunity (ETI) (Chisholm et al., 2006; Jones
and Dangl, 2006). PTI is a first line of defense activated upon
recognition ofMAMPs (microbe-associatedmolecular patterns) or
PAMPs, which are ubiquitous structural elements of molecules
essential to themicrobial lifestyle. Perception of PAMPsbypattern
recognition receptors (PRRs) leads to the elicitation of PTI. The
prototype PRR in Arabidopsis thaliana is FLS2, which is the
receptor for a polypeptide PAMP (flg22) present in the flagellin
protein of some bacteria (Felix et al., 1999). PTI from activated
FLS2 and other PRRs includes generation of reactive oxygen
species (ROS), activation of MAP kinases, production of the plant
hormones SA and ethylene, transcriptional reprogramming, and
cell wall fortificationmarkedby callose deposition (Chinchilla et al.,
2007; Tsuda et al., 2008). Various PAMPS, including flg22, Ef-Tu,
and chitin, induce activation of a common set of genes, indicating
that signals from PRRsmay converge on a limited set of pathways
(Zipfel et al., 2006;Wan et al., 2008). PTI typically does not cause a
hypersensitive response (HR) or localized cell death, but none-
theless effectively combats potentially pathogenic microbes.
Pathogens counter this first line of defense by delivering PTI-
suppressing virulence effectors. Suppression of PTI is hypothe-
sized to be a key step in the evolution of pathogenicity and can
lead to a diseased state also known as effector-triggered
susceptibility (ETS) (Jones and Dangl, 2006). Gram-negative bac-
teria use type III secretion systems (T3Ss) to deliver defense-
suppressing type III effectors (T3Es) into the cytosol of plant cells
(Alfano and Collmer, 2004). Individual bacteria deliver a repertoire
of T3Es that move to a variety of subcellular locations and perturb
various host targets (da Cunha et al., 2007).Many T3Es have been
shown to suppress PTI, including limiting callose deposition and
enhancing the growth of T3S-deficient bacteria (Guo et al., 2009).
To counter ETS, R-genes mediate recognition of pathogen-
encoded effectors and activate ETI. The prototypical R-proteins
are composed of a central nucleotide binding site and C-terminal
leucine-rich repeats (McHale et al., 2006). These intracellular
R-proteins function like receptors that either interact with effec-
tors directly or perceive effectors indirectly via their perturbations
of host targets (Mackey and McFall, 2006). ETI typically pro-
duces a robust defense response that potently restricts the
growth of microbes and frequently elicits a HR. Current data
support models in which differences between the outputs of ETI
and PTI are quantitative rather than qualitative (Maleck et al.,
2000; Tao et al., 2003; Jones and Dangl, 2006; Shen et al., 2007).
RIN4 (RPM1-interacting protein 4) is a multifunctional protein
that links PTI, ETS, and ETI. RIN4 is a negative regulator of PTI
(Kim et al., 2005b). Arabidopsis plants lacking or inducibly ex-
pressing RIN4 display enhanced or suppressed flg22-induced
callosedeposition, respectively.Pseudomonas syringaepv tomato
1 These authors contributed equally to this work.2 Current address: Universidade Federal de Vicosa, Departamento deFitopatologia, Campus Universitario Vicosa, Minas Gerais, Brazil 36570-000.3 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: David Mackey([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.111.088708
The Plant Cell, Vol. 23: 3798–3811, October 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved.
strain DC3000 (PstDC3000) is pathogenic on tomato and Arabi-
dopsis, but the T3S-deficient hrcCmutant fails to grow, because it
is unable to deliver PTI-suppressing T3Es (Hauck et al., 2003).
Thus, the ability of hrcC to grow is a useful proxy for suppression of
PTI. The hrcC mutant grows to reduced or elevated levels in
Arabidopsis plants lacking or inducibly expressing RIN4, respec-
tively.
Numerous T3Es target RIN4 as part of their attempt to cause
ETS. The T3Es AvrRpm1, AvrB, and AvrRpt2 each suppress
flg22-induced callose deposition and promote the growth of
hrcC (Kim et al., 2005b; Shang et al., 2006). AvrRpm1 and AvrB
each induce phosphorylation of RIN4, whereas AvrRpt2 proteo-
lytically clips RIN4 into three pieces (Mackey et al., 2002; Axtell
et al., 2003; Mackey et al., 2003; Chisholm et al., 2005; Kim et al.,
2005a; Takemoto and Jones, 2005). RPM1-induced protein
kinase (RIPK) is a receptor-like cytoplasmic kinase that contrib-
utes toT3E-induced phosphorylation of RIN4 (Liu et al., 2011). It
has proven difficult to determine how perturbation of RIN4 by
AvrRpm1, AvrB, and AvrRpt2 regulates the PTI-suppressing
function of RIN4, because each of these T3Es has additional
virulence targets inside plant cells (Belkhadir et al., 2004; Lim and
Kunkel, 2004). Targeting of RIN4 by HopF2, a T3E with ADP-
ribosyltransferase activity, is required for its virulence activity in
Arabidopsis, further demonstrating the bona fide role of RIN4 in
defense regulation (Wang et al., 2010; Wilton et al., 2010).
Perturbation of RIN4 by T3Es mediates the induction of ETI by
two Arabidopsis R-proteins, RPM1 and RPS2. Despite lacking
predicted transmembrane domains, RIN4, RPM1, and RPS2
each localize to the plasma membrane (Boyes et al., 1998; Axtell
and Staskawicz, 2003; Takemoto and Jones, 2005). Palmitoyla-
tion of C-terminal cysteines mediates membrane anchoring of
RIN4 (Kim et al., 2005a). RIN4 interacts with both RPM1 and
RPS2, perhaps targeting them to the membrane, and prevents
ectopic activation of both R-proteins (Mackey et al., 2002; Axtell
and Staskawicz, 2003; Mackey et al., 2003; Belkhadir et al.,
2004; Day et al., 2005). RPM1 responds strongly to AvrRpm1 and
AvrB andweakly to AvrRpt2, whereas RPS2 responds strongly to
AvrRpt2 and weakly to AvrRpm1 and AvrB (Kim et al., 2009). The
activation of RPM1 by AvrB, and partially by AvrRpm1, is
mediated through the phosphorylation of amino acid T166, a
residue targeted by RIPK (Chung et al., 2011; Liu et al., 2011).
Thus, each of these R-proteins seems to induce defense re-
sponses of different strengths in response to different perturba-
tions of RIN4. In addition to these PTI-, ETS-, and ETI-related
functions of RIN4 that are manifested when bacteria invade the
apoplast of the plant leaf, RIN4 also negatively regulates prein-
vasion defense via interaction with (H+)–ATPases that promote
reopening of stomata after perception of bacterial PAMPs (Liu
et al., 2009).
We performed a structure–function analysis of postinvasion
defense regulation by RIN4. We show that separable fragments
of RIN4, each containing a plant-specific nitrate-induced (NOI)
domain (Pfam: PF05627), suppress PTI. Notably, these results
include two derivatives of RIN4 that correspond to the two
primary AvrRpt2 cleavage products (ACP2 and ACP3) of RIN4.
We show that separable fragments, each containing an NOI
domain, contribute to PTI suppression by RIN4. Phylogenetic
analysis indicates that distinct N-terminal NOI (N-NOI) and
C-terminal NOI (C-NOI) domains have been maintained in RIN4
homologs across the plant lineages. We show that RIN4 deriv-
atives that are not properly targeted to the plasma membrane,
because they lack the C-terminal region or the cysteines in the
C-terminal region, elicit a cell death response. In a triple mutant
background that prevents the cell death response, we demon-
strate that nonmembrane-tethered derivatives are more potent
suppressors of PTI, indicating that membrane targeting of RIN4
limits its ability to suppress PTI and that AvrRpt2 may enhance
PTI suppression by liberating a hyperactive fragment of RIN4
from the membrane. Collectively, our results indicate that both
NOI-containing fragments of RIN4 contribute to negative regu-
lation of PTI and that cleavage of RIN4 by AvrRpt2 produces two
active PTI-suppressing fragments, including one that is no longer
tethered to the plasma membrane.
RESULTS
RIN4 Contains Separable Domains Capable of
Suppressing PTI
Our initial goal was to identify the protein region(s) of RIN4 that
mediate suppression of PTI. To this end, we generated trans-
genic Arabidopsis lines that inducibly express RIN4 derivatives,
including the N-terminal two thirds (149D211), C-terminal one
third (1D141), and the two major AvrRpt2 proteolytic products
consisting of AA 11 to 152 (ACP2) and AA 153 to 211 (ACP3) in
wild-type Columbia-0 ecotype (Col-0) plants (Figure 1A). All the
RIN4 derivatives in Figure 1 and throughout this study, including
full-length RIN4 (RIN4FL), have an N-terminal T7 tag to permit
uniform assessment of their expression. Also, for all derivatives,
two or more independent transgenic lines with single homozy-
gous insertions were produced and tested, and data from single
representative lines are presented.
The effect of inducibly expressed RIN4 derivatives on PTI
signaling was tested in two assays: callose deposition in re-
sponse to flg22 infiltration and growth of the hrcC mutant of
PstDC3000. Expression of the RIN4 derivatives was induced by
spraying 4- to 5-week-old plants with dexamethasone (Dex). At
48 h after induction, leaves were syringe-infiltrated with either 30
mM flg22 or 105 colony-forming units (CFU) per mL of hrcC
bacterial suspension. Callose deposition was measured by an-
iline blue staining at 16 h after flg22 infiltration (Figure 1B). Growth
of hrcC bacteria was measured at 4 d postinfiltration (Figure 1C).
Similar to RIN4FL, the N-terminal fragments of RIN4 (ACP2 or
149D211) potently suppressed callose deposition and enhanced
the growth of hrcC. The C-terminal fragments of RIN4 (ACP3 or
1D141) failed to suppress flg22-induced callose deposition but
still permitted the hrcCmutant to grow to high levels. Thus, these
derivatives are all capable of suppressing effective defense
against hrcC, but only the N-terminal fragments suppressed
flg22-induced callose deposition.
Immunoblotting was used to assess the expression of the
RIN4 derivatives (Figure 1D). Immunoblots detecting the T7
epitope were used to compare expression levels of the deriva-
tives relative to one another, and blots detecting RIN4 were used
to compare expression levels of RIN4FL with that of native RIN4
Suppression of PTI by RIN4 3799
in nontransgenic Col-0. The anti-RIN4 blot shows that the overall
level of RIN4 protein (native RIN4 plus RIN4FL) in the RIN4FL line
was only modestly elevated (approximately twofold) relative to
the level of RIN4 in Col-0. Although the ACP3 and 149D211
derivatives accumulated to only ;20% the levels of RIN4FL,
each of these derivatives was able to suppress PTI. We also
examined the accumulation of ACP2 and ACP3 from native RIN4
after delivery of AvrRpt2 during P. syringae infection (see Sup-
plemental Figure 1 online). ACP3 accumulated detectably and
remained membrane-associated, consistent with a previous
report (Kim et al., 2005a). ACP2 is only weakly detected by our
RIN4 polyclonal sera, because the major epitope(s) are in the
C-NOI. Despite this limitation, we detected accumulation of
ACP2 released from native RIN4 by AvrRpt2 at 3.5 and 6 h after
bacterial infiltration (see Supplemental Figure 1 online). Because
PAMP-induced responses occur rapidly (e.g., even the “late”
induction of callose by flg22 is apparent by 4 h after infiltration
[Kim et al., 2005b]), the ACP2 and ACP3 fragments released by
AvrRpt2 from native RIN4 persist long enough to suppress PTI.
Fragments Containing N- and C-NOI Regions Redundantly
Contribute to PTI Suppression by RIN4
Figure 1 showed that separable fragments of RIN4 are individ-
ually capable of suppressing PTI. Because each of the deriva-
tives tested in Figure 1 contains a plant-specific NOI domain
(Figure 1A), we speculated that the NOI domains contribute to
PTI suppression by RIN4. To determine the role of the N-NOI and
C-NOI, we constructed lines expressing RIN4 derivatives lacking
N-NOI (1D64), C-NOI (149D176), or both (1D64 and 149D176 =
DDNOI) (Figure 2A). The 1D64 and 149D176 derivatives, each
with one NOI, retained the ability to suppress flg22-induced
Figure 1. RIN4 Contains Separable Domains with Distinct Defense Suppressing Activities.
(A) Schematic diagram of the 211 amino acid RIN4 protein and its derivatives. RIN4 contains two conserved NOI domains (black bars), two AvrRpt2
cleavage sites (triangles), and C-terminal cysteines required for acylation and membrane targeting of RIN4 (CCC, amino acids 203, 204, and 205). Full-
length RIN4 (RIN4FL) and other derivatives all contain an N-terminal T7-tag (black rectangle). AvrRpt2 cleaves RIN4 between amino acids 10/11 and
152/153 and the derivatives ACP2 and ACP3 (AvrRpt2-cleavage products) consist of RIN4 amino acids 11 to 152 and 153 to 211, respectively. w.t., wild
type.
(B) and (C)Callose induced by flg22 infiltration (B) and growth of hrcC (C) in Col-0 and lines of Col-0 inducibly expressing the indicated RIN4 derivatives.
Expression was induced with 20 mM dexamethasone 48 h before infiltration with (B) 30 mM flg22 or (C) 1 3 105 CFU/mL of hrcC. Numbers in (B) are
average and SD of the number of callose deposits per 1.1 mm2. Bacterial numbers and SD (C) are plotted at the time of infiltration (day 0) in Col-0 and
after 4 d. *P = 0.008; **P < 0.0002 [two-tailed Student’s t test for comparison with Col-0 plants].
(D) Immunoblots with anti-T7 antisera to compare levels of RIN4 derivatives and with anti-RIN4 antisera to compare levels of native RIN4 and RIN4FL.
Numbers are band intensities with the signal in plants expressing RIN4FL normalized to 1. Panels below are ponceau stains for total protein loading.
3800 The Plant Cell
callose deposition (Figure 2B) and to promote growth of the hrcC
mutant (Figure 2C). On the contrary, theDDNOI derivative lacking
both NOIs was unable to suppress PTI in either assay (Figures 2B
and 2C). The inability ofDDNOI to suppress PTI was not the result
of poor expression of this derivative (Figure 2D), but might result
from disrupted protein structure. The ability of 149D176 to
promote growth of hrcC was variable between assays, possibly
because of the low expression level of this derivative. Figure 2C
shows an example of those experiments in which this derivative
was as active as RIN4FL, whereas in other experiments, it
supported growth of hrcC to levels intermediate betweenRIN4FL
and the nontransgenic Col-0 plants.
TheN- andC-terminal fragments of RIN4 differ in their ability to
suppress flg22-induced callose deposition (Figure 1). Derivatives
lacking either NOI individually (1D64 and 149D176) suppress
flg22-induced callose deposition, so differences between the
NOI domains are not sufficient to account for differences in
suppression of callose (Figure 2). One possible explanation is
that sequences located between the two NOIs, which are part of
the N-terminal fragments (149D211 and ACP2), are required for
suppressing flg22-induced callose deposition. To examine this
possibility, we tested the PTI suppression of RIN4 derivatives
lacking residues between the NOIs (65D91, 94D111, and
112D141) (see Supplemental Figure 2A online). In these deriva-
tives, all of the residues from AA 65 to AA 141 except two Gly (AA
92 and 93) were deleted; however, each derivative was able to
suppress callose deposition and enhance growth of hrcC relative
to nontransgenic Col-0 plants (see Supplemental Figures 2B and
2C online). Thus, none of these intervening sequences are
essential for suppression of PTI. All three internal deletion deriv-
atives were inducibly expressed and membrane-localized as
predicted (see Supplemental Figure 3 online). Growth of hrcC in
plants expressing 65D91 was intermediate between Col-0 plants
and plants expressing RIN4FL, possibly because of the low
expression level of 65D91 (see Supplemental Figure 3 online).
Because sequences between the NOIs were not essential for
suppression of callose deposition, an alternative explanation for
the difference between the N- and C-terminal derivatives is that
the N-terminal derivatives are not membrane-associated (see
Supplemental Figure 3 online), which enhances the defense-
suppressing activity of RIN4 derivatives (see below).
The N- and C-NOIs Are Evolutionarily Distinct
Based on the hypothesized role of theN-NOI andC-NOI domains
in the suppression of PTI (Figure 2), we took a phylogenetic
approach to examine the relationship of the NOIs from RIN4,
other NOI-containing proteins of Arabidopsis, and the closest
RIN4 homologs (by protein Blast) from various plant lineages,
including several monocots and moss. The NOI sequences,
starting with the AvrRpt2 cleavage sites (Chisholm et al., 2005)
were aligned by ClustalW (see Supplemental Figure 4 and
Figure 2. NOI Domains Are Required for Defense-Suppressing Activity of RIN4.
(A) Schematic diagram of RIN4 derivatives as in Figure 1. DDNOI lacks amino acids 1 to 64 and 149 to 176. w.t., wild type.
(B) and (C) Callose induced by flg22 infiltration (B) and growth of hrcC (C) in Col-0 and lines of Col-0 inducibly expressing the indicated RIN4 derivatives
as in Figure 1. *P < 0.005; **P < 0.05 [two-tailed Student’s t test for comparison with Col-0 plants].
(D) Immunoblots with anti-T7 antisera to compare levels of RIN4 derivatives as in Figure 1.
Suppression of PTI by RIN4 3801
Supplemental Data Set 1 online). Only two of the examined NOIs,
theC-NOIs of theArabidopsis proteins NOI10 (At5G48657.2) and
NOI11 (At3G07195.1), lack predicted cleavage sites for AvrRpt2.
Based on this alignment, the evolutionary history of the NOI
domains was inferred by the neighbor-joining method (Figure 3).
Remarkably, the N-NOI domains (red squares) and C-NOI do-
mains (blue diamonds) of RIN4 homologs clustered into separate
clades, indicating that the two domains have remained distinct
since before the divergence of moss and vascular plants (;400
million years ago). In addition to amino acid sequence differ-
ences, a prominent distinguishing feature betweenNOIs from the
two clades is the difference in spacing between the AvrRpt2
cleavage site and the more C-terminal–conserved residues (see
Supplemental Figure 4 online). The distinctness of the two clades
is even more pronounced than what is represented in Figure 3,
because the tree was constructed using the complete deletion
option, in which all positions containing gaps and missing data,
including the spacing difference, were eliminated from the data
set. Thus, although both contribute to PTI suppression by RIN4,
the N-NOI and C-NOI are evolutionarily and perhaps functionally
distinct.
The NOI domains of other NOI-containing proteins from
Arabidopsis fall into both clades (Figure 3). Two of these NOI-
proteins (NOI10 and NOI11) have domain architectures similar to
RIN4 with N- and C-NOI domains. The C-NOIs of NOI10 and
NOI11 lack predicted AvrRpt2 cleavage sites. The N-NOIs (or-
ange squares) and C-NOIs (purple diamonds) of NOI10 and
NOI11 are in the same clades as the N-NOI and C-NOI of RIN4,
respectively. The other Arabidopsis proteins contain single NOIs
(black squares in Figure 3) that, with the exception of NOI9, are in
the C-NOI clade.
Structure of the N Terminus Regulates PTI Suppression
by RIN4
An N-terminal deletion (1D31) disrupted the PTI-suppressing
function of RIN4; the 1D31 derivative failed to suppress flg22-
induced callose deposition or promote the growth of hrcC (see
Supplemental Figure 5 online). Specific residues in the first 31
amino acids of RIN4 are not essential for PTI suppression,
because the 1D64 derivative retained the ability to suppress PTI
(Figure 2; see Supplemental Figure 5 online). Expression levels
do not account for the difference, because 1D31 and 1D64
accumulated to similar levels (see Supplemental Figure 5D
online). Interestingly, the 1D31 derivative retains some activity.
When expressed as a transgene under control of the native RIN4
promoter in rpm1 rps2 rin4RPM1-myc plants (Boyes et al., 1998;
Belkhadir et al., 2004), 1D31 restored RPM1-myc–mediated HR
in response to AvrRpm1 or AvrB (see Supplemental Figure 6
online). Using the same expression method, we have shown that
1D141 supports RPM1 function and that the C-NOI (AA 149 to
176) is required for RPM1 function (Chung et al., 2011).
Nonmembrane-Tethered Derivatives of RIN4 Elicit Cell
Death in Col-0
Arabidopsis RIN4 has three cysteines (AA 203, 204, and 205)
near its C terminus that mediate palmitoylation and membrane
targeting of the protein and that are key for its ability to suppress
RPS2 activity (Day et al., 2005; Kim et al., 2005a). We looked for
the presence of similar sequences in the RIN4 homologs and
Arabidopsis NOI-containing proteins from Figure 3. Remarkably,
all of these proteins have one to three cysteineswithin the final 12
AA of their C termini, and the cysteines are closely preceded and/
or followed by aromatic AAs, especially phenylalanines (see
Supplemental Figure 7 online). The phenylalanines following the
three cysteines at the C terminus of RIN4 were shown to be
essential for membrane localization (Takemoto and Jones,
2005). These data indicate that most or all of these NOI-
containing proteins are likely acylated and membrane-tethered
through a process similar to that which targets RIN4 (Kim et al.,
2005a). Thus, membrane localization may be generally relevant
to the functions of NOI-containing proteins.
To examine how membrane tethering affects the function of
RIN4, we examined RIN4 derivatives with mutations in the
C-terminal region that disrupt the acylation site (177D211 and
CCC>AAA) (Figure 4A). Expression of 177D211 and CCC>AAA
elicited PR-1 expression and amacroscopic cell death response
in Col-0 (Figures 4B and 4C). Trypan blue staining revealed that
RIN4FL also induces cell death in Col-0, albeit to a lesser extent
than 177D211 and CCC>AAA (Figure 4D). The cell death phe-
notype observed was not a result of these derivatives being
expressed to a higher level than RIN4FL. In fact, consistent with
earlier results (Kim et al., 2005a), 177D211 and CCC>AAA
accumulated to significantly lower levels than RIN4FL in Col-0
(Figure 4E; see Supplemental Figure 3 online). Subcellular frac-
tionation confirmed that all of the derivatives predicted to be
nonmembrane-tethered were in the soluble fraction as predicted
(Figure 4F; see Supplemental Figure 3 online).
We sought to test the PTI-suppressing activity of nonmem-
brane-tethered derivatives of RIN4 in the absence of cell death.
We hypothesized that the defense and cell death response
elicited by expression of nonmembrane-tethered RIN4 deriva-
tives was the result of activation of RPM1 and/or RPS2 or
of interaction with native RIN4. Thus, we constructed new
transgenic lines that inducibly express RIN4FL, 177D211, or
CCC>AAA in the rpm1 rps2 rin4 triplemutant background. Unlike
in Col-0, expression of RIN4FL, 177D211, andCCC>AAA caused
no cell death response in the triple mutant (Figures 4C and 4D).
This result does not distinguish between whether the cell death
induced by nonmembrane-tethered derivatives of RIN4 in Col-0
is dependent on RIN4, RPM1, and/or RPS2.
Nonmembrane-Tethered Derivatives Are Hyperactive
Suppressors of PTI
Cleavage of RIN4 by AvrRpt2 has been equated to disappear-
ance of RIN4 (Mackey et al., 2003; Day et al., 2005; Kim et al.,
2005a; Kim et al., 2005b). However, it is difficult to explain why a
T3E would eliminate a negative regulator of PTI. We hypothe-
sized that AvrRpt2 does not simply eliminate RIN4, but instead
produces fragments of RIN4 that actively suppress PTI. Indeed,
ACP2 andACP3,which accumulate after cleavage of native RIN4
by AvrRpt2 (see Supplemental Figure 1 online), were each
capable of potently suppressing PTI when inducibly expressed
(Figure 1).
3802 The Plant Cell
Figure 3. The N-NOI and C-NOI of RIN4 Are from Evolutionarily Distinct Clades.
The NOI domains from RIN4, its closest BLAST homolog from a variety of plants, and other NOI-containing proteins from Arabidopsis (NOI1 to NOI14)
were analyzed. The NOI domains were aligned by ClustalW, and their evolutionary relationship was inferred by the Neighbor-Joining method using
MEGA4. The confidence probability (3100) that the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates), is
shown next to the branches. Branch lengths represent evolutionary distances. Number ranges are the amino acid positions of each NOI within its
respective protein. The N-NOIs and C-NOIs from RIN4 homologs from different plant species are indicated by red squares and blue diamonds,
respectively. Orange squares and purple diamonds indicate the N-NOIs and C-NOIs from Arabidopsis NOI proteins with a structure similar to RIN4
(NOI10 and NOI11). Black triangles indicate the NOIs from Arabidopsis proteins with only a single NOI domain. All NOIs start with an AvrRpt2 cleavage
site, with the exception of the C-NOIs of NOI10 and NOI11 from Arabidopsis, which have a Pro insertion and Trp substitution predicted to disrupt
cleavage by AvrRpt2. NOI8 (At5G18310) is excluded from the analysis, because ambiguous gene models exist in TAIR9. RIN4 is At3G25070. For other
Arabidopsis proteins containing NOIs, the AGI numbers are shown. RIN4 homologs are from the following plant species: A. lyrata, B. juncea, M.
truncatula, L. saligna, S. lycopersicum, P. trichocarpa, V. vinifera, O. sativa, Z. mays, B. distachyon, and P. patens.
Suppression of PTI by RIN4 3803
Using the lines expressing RIN4FL, 177D211, and CCC>AAA
in the rpm1 rps2 rin4 background, we compared the ability of
these nonmembrane-tethered RIN4 derivatives to suppress PTI
in the absence of cell death. First, we examined their ability to
suppress flg22-induced callose deposition (Figure 5A). SALK
T-DNA insertions, including the rin4 mutation, cause frequent
posttranscriptional gene silencing of the Dex responsive tran-
scription factor in the Dex-inducible system (Geng and Mackey,
2011). We took advantage of this fact to examine the capacity of
RIN4 derivatives to suppress callose deposition when expressed
at low levels, which revealed differences in the activity of the
individual derivatives. Multiple plants for each line were grown,
sprayed with Dex, and subsequently infiltrated with flg22. In
addition to processing the flg22-infiltrated leaves for callose
staining, noninfiltrated leaves from the same plants were har-
vested for anti-T7 immunoblotting. The results of the immuno-
blots were used to select individual plants with comparable, low
levels of expression of RIN4FL, 177D211, and CCC>AAA (see
Supplemental Figure 8 online). Among the selected plants, those
expressing the nonmembrane-tethered derivatives of RIN4
produced fewer flg22-induced callose deposits than those ex-
pressing RIN4FL (Figure 5A). Figure 1 shows that nonmembrane-
tethered derivatives of RIN4 suppress flg22-induced callose
deposition as well as RIN4FL when expressed at much lower
levels. Figure 5A shows that the derivatives suppress callose
depositionmore efficiently thanRIN4FLwhen the derivatives and
RIN4FL are expressed at comparable low levels.
Nonmembrane-tethered derivatives of RIN4 also potently pro-
mote the growth of hrcC. In Col-0 plants, expression of 177D211
caused hrcC to grow to a level two orders of magnitude higher
than did RIN4FL (Figure 5B), despite the fact that it does
not accumulate as well (Figure 4E). Col-0 plants expressing
CCC>AAA were not included, because they undergo rapid cell
death within 36 h of Dex spray. To assess the effect of inducible
expression of RIN4 derivatives in the absence of cell death, we
used the lines in rpm1 rps2 rin4 plants (Figure 5B). RIN4FL did
not enhance the growth of hrcC in the rpm1 rps2 rin4 triple mu-
tant background, possibly because of low levels of expression
Figure 4. Nonmembrane-Tethered Derivatives of RIN4 Cause a Cell Death Response in Col-0.
(A) Schematic representation of RIN4FL and derivatives as in Figure 1. CCC>AAA has RIN4 Cys203/204/205 substituted by Ala203/204/205. w.t., wild
type.
(B) Immunoblot showing PR-1 expression in Col-0 and transgenic plants expressing RIN4FL, CCC>AAA, and 177D211 at 48 h after Dex treatment.
(C)CCC>AAA and 177D211 cause a cell death–like response in Col-0 but not in rpm1 rps2 rin4. Pictures show plant phenotypes 72 h after RIN4FL or the
indicated derivatives were induced by Dex in either Col-0 or rpm1 rps2 rin4. Macroscopic cell death was evident in plants expressing either CCC>AAA
or 177D211 in Col-0, whereas mild symptoms were observed in Col-0 plants expressing RIN4FL. Cell death symptoms were not observed for any of the
RIN4 derivatives in rpm1 rps2 rin4 plants.
(D) Trypan blue staining of leaves from plants shown in (C). Leaves of plants were collected 72 h after Dex spray and were stained with trypan blue.
Consistent with results obtained in (C), CCC>AAA and 177D211 show strong cell death in Col-0 but not in rpm1 rps2 rin4.
(E) Anti-T7 immunoblot showing that CCC>AAA and 177D211 accumulate to similar levels in Col-0 and rpm1 rps2 rin4 plants 48 h after mock (�) or Dex
(+) spray. Col-0 plants expressing the CCC>AAA derivative are not included, because the tissue is completely collapsed by 48 h.
(F) Total protein extracts (T) of Col-0 leaves expressing indicated derivatives of RIN4 were separated into soluble (S) and microsomal (M) fractions and
were subjected to anti-T7 immunoblotting. The microsomal fractions are overloaded by approximately fivefold relative to the total and soluble fractions.
Ponceau red staining (shown below T7 blots) demonstrates that RuBisCo partitions into the soluble fraction.
3804 The Plant Cell
resulting from posttranscriptional gene silencing. Despite similar
low levels of expression (see Supplemental Figure 9 online),
177D211 and CCC>AAA still enabled hrcC to grow to high levels.
Collectively, the growth and callose data in Figure 5 indicate that
inducibly expressed RIN4 more potently suppresses PTI when it
is not tethered to the plasma membrane.
To learn more about the PTI-suppressing activity of RIN4FL
and nonmembrane-tethered derivatives of RIN4, we examined
the effect of RIN4FL, 177D211, and CCC>AAA on early PTI
responses (see Supplemental Figures 10 and 11 online). The
accumulation of reactive oxygen is a very early response to flg22
(Felix et al., 1999) that depends on ethylene signaling (Mersmann
et al., 2010). We used a luminol-based assay to examine the
induction of ROS induced by flg22. To avoid ROS production
associated with cell death in Col-0, we used the lines expressing
these derivatives in the rpm1 rps2 rin4 triplemutant. In addition to
Arabidopsis plants expressing RIN4 derivatives, rpm1 rps2 rin4
plants were included as a positive control, and fls2 plants were
included as a negative control. After measuring flg22-induced
ROS, immunoblots were done to examine plants for expression
levels of the RIN4 derivatives (see Supplemental Figure 9 online).
Traces from selected plants were then used to determine the
peak value of ROS accumulation (sample traces from individual
plants are shown in Supplemental Figure 10A online), and data
from five biological replicates were normalized with the average
peak values in rpm1 rps2 rin4 set to one and combined (see
Supplemental Figure 10B online). Although slight decreases in
the peak accumulation of ROS relative to rpm1 rps2 rin4 were
observed with the RIN4 derivatives, these decreases were of a
much lower magnitude than the decrease in flg22-induced
callose deposition in the same plants (Figure 5). Furthermore,
the decreases in ROS in plants expressing RIN4 derivatives were
not significant relative to the rpm1 rps2 rin4 control plants
(two-tailed t test P values for all were >0.07). Thus, inducible
expression of RIN4 derivatives, including the potent nonmem-
brane-tethered derivatives, did not efficiently suppress PTI-
induced ROS accumulation.
We also tested the effect of RIN4 derivatives on early, PTI-
induced gene expression. Leaves of plants induced to express
RIN4FL, 177D211, and the inactive DDNOI, as well as Col-0 and
fls2 control plants, were infiltrated with flg22 or H20. After 2 h,
quantitative real-time PCR was used to measure FRK1 and
MYB51 transcript levels (see Supplemental Figure 11 online). In
Col-0 plants and DDNOI-expressing plants, each transcript
accumulated to higher levels after infiltration with flg22 than
with water. Each transcript remained expressed at low levels
after flg22 infiltration into fls2 plants. These findings confirm that
both genes are flg22-induced. In plants expressing RIN4FL,
MYB51 expression was elevated in water-infiltrated plants, and
expression of both FRK1 and MYB51 were induced by flg22
relative to water. In plants expressing 177D211, expression of
both genes was elevated in water-infiltrated plants, and no
further increase was observed in flg22-infiltrated plants. Collec-
tively, these data indicate that RIN4FL fails to suppress rapid PTI-
induction of ROS and gene expression. Similar to RIN4FL, the
nonmembrane-tethered 177D211 derivative fails to suppress
ROS. However, in plants expressing 177D211, flg22 did not
induce expression of FRK1 and MYB51 above their already
elevated levels.
DISCUSSION
To better understand how RIN4 links ETI, ETS, and PTI, we
performed a structure–function analysis of postinvasion defense
regulation by RIN4 (data summarized in Supplemental Figure
12 online). We show that separable fragments of RIN4—the
N-terminal two thirds and the C-terminal one third of the protein,
including two derivatives of RIN4 that correspond to the two
primary AvrRpt2 cleavage products (ACP2 and ACP3)—are
capable of suppressing PTI. Notably, ACP2 and ACP3 can be
detected from native RIN4 after cleavage by bacterially delivered
AvrRpt2. Thus, RIN4 contains nonoverlapping fragments with
Figure 5. Derivatives of RIN4 Suppress PTI More Potently When Not
Membrane-Tethered.
(A) Expression of RIN4 derivatives in rpm1 rps2 rin4 plants was induced
by spraying with Dex 48 h before infiltration with 30 mM flg22. Callose
numbers were counted from plants expressing comparable levels of
RIN4FL, CCC>AAA, and 177D211 proteins (see Supplemental Figure 8
online). The results shown are averaged from three independent biolog-
ical replicates, with the average number of callose deposits induced in
plants expressing RIN4FL in each experiment normalized to 1. Error bars
represent SD. *, P = 0.001; **P < 0.0001 [two-tailed Student’s t test for
comparison with rpm1 rps2 rin4 plants expressing RIN4FL].
(B) Expression of RIN4 derivatives in Col-0 or rpm1 rps2 rin4 plants was
induced by spraying with Dex 48 h before infiltration with 105 CFU/mL of
hrcC. Growth data in rpm1 rps2 rin4 was obtained from plants express-
ing similar, low levels of RIN4FL and derivatives (see Supplemental
Figure 9 online). Numbers of hrcCwere determined 0 (gray bars), 2 (white
bars), and 4 (black bars) d after infiltration. Error bars represent SD. *P <
0.05 [two-tailed t test for comparison with rpm1 rps2 rin4 plants]; **P <
0.005 [two-tailed Student’s t test for pairwise comparison with Col-0
plants].
Suppression of PTI by RIN4 3805
defense-suppressing activity, each containing a plant-specific
NOI domain. The N-NOI and C-NOI domains of RIN4 belong to
evolutionary clades that have remained distinct from each other
since ;400 million years ago. However, at the level of our
assays, these domains contribute redundantly to defense sup-
pression; derivatives lacking either the N-NOI or C-NOI (1D64 or
149D176) effectively suppress callose deposition and promote
the growth of T3S-deficient bacteria, whereas a derivative
lacking both is inactive. Nonetheless, it seems likely that the
N-NOI and C-NOI will differ in their function. Indeed, the C-NOI
includes a fragment of RIN4 that interacts with AvrB (Desveaux
et al., 2007) and is required for AvrRpm1- and AvrB-induced
activation of RPM1(Chung et al., 2011; Liu et al., 2011). The
Arabidopsis genome encodes two proteins that, similar to RIN4,
have two NOI domains and 12 proteins with a single NOI domain.
The importance of the NOIs for defense suppression by RIN4
highlights the potential role of these other NOI-containing pro-
teins in regulating plant defense.
N- and C-terminal fragments of RIN4 differ in their ability to
suppress callose deposition. Both 1D141 and ACP3 promote the
growth of hrcC but are unable to suppress callose deposition.
This is in contrast with 149D211 and ACP2, which promote the
growth of hrcC and suppress callose deposition. Several possi-
ble explanations might account for this difference between the
N- and C-terminal fragments of RIN4. One is that the threshold
for suppressing callose deposition is more stringent than the
threshold for promoting growth of the hrcC mutant bacteria. A
second possible explanation is a difference in the activity of the
N-NOI and C-NOI domains of RIN4. However, derivatives of
RIN4 lacking either NOI (1D64 or 149D176) retain the ability to
suppress callose as well as to promote growth of T3S-deficient
bacteria. A third possible explanation is that there is a function for
the sequences between the NOIs that are included in 149D211
and ACP2 and are absent in 1D141 and ACP3. Although these
intervening sequences do not contain any residues absolutely
required to suppress callose deposition, their absence might
affect RIN4 function through structural perturbation. A fourth
possible explanation to account for the difference in PTI sup-
pression between N- and C-terminal fragments of RIN4 is that
the N-terminal fragments are not tethered to the plasma mem-
brane, which enhances their PTI-suppressing activity.
The subcellular localization of RIN4 dramatically affects its
activity. Derivatives of RIN4 lacking the C-terminal cysteines are
no longer membrane-tethered. In Col-0, inducible expression of
these nonmembrane-tethered derivatives causes a cell death
response. Derivatives lacking the C terminus are also potent
suppressors of PTI in Col-0. However, it is unclear how the cell
death response affects our PTI readouts. To circumvent this
problem, we expressed nonmembrane-tethered derivatives in
an rpm1 rps2 rin4 triple mutant and observed that they no longer
caused cell death. In this background, the nonmembrane-teth-
ered derivatives suppress PTI. In fact, assays for growth of hrcC
mutant bacteria and callose deposition revealed that these
derivatives suppress PTI more potently than does RIN4FL.
Thus, anchoring of RIN4 to the plasma membrane limits its
defense-suppressing capacity. Release of RIN4 via cleavage by
AvrRpt2, or by an endogenous protease (Luo et al., 2009), could
thus release a hyperactive defense-suppressing fragment of
RIN4. Notably, the nonmembrane-tethered derivative 177D211
prevents flg22 from further elevating expression of FRK1 and
MYB51. However, interpretation of this result is difficult, because
expression of both genes is already elevated in plants expressing
177D211. Release from the plasma membrane could enhance
the ability of fragments of RIN4 to suppress defense transcription
downstream of cytoplasmic and nuclear localized MPK4 (Cui
et al., 2010). Further work is necessary to determine the subcel-
lular location and mode of action by which the nonmembrane-
tethered derivatives act to promote cell death and suppress PTI.
A structural model generated by the I-TASSER server (http://
zhanglab.ccmb.med.umich.edu/I-TASSER/) on the basis of mul-
tiple-threading alignments by LOMETS and iterative TASSER
simulations (Zhang, 2008) predicted a globular structure for RIN4
(see Supplemental Figure 13 online). In addition to the I-TASSER
model, modeling RIN4FL using four other top-ranked prediction
servers in the CASP8 and CASP9 experiments also yielded
globular structures (see Supplemental Figure 14 online). The
robustness of the I-TASSER model was supported by Rama-
chandran plot (see Supplemental Figure 15 online), in which
96.7% of the residues were in favored and allowed regions, and
only 3.3% of residues were in disallowed regions (Morris et al.,
1992). We favor the I-TASSER model, because it fits with
available data regarding the structure and function of RIN4 and
because I-TASSER was the top-ranked server in the CASP7,
CASP8, and CASP9 experiments. First, the N-terminal two thirds
and the C-terminal one third of the protein fold into separate
domains (see Supplemental Figure 13A online), consistent with
the functionality of each of these domains in isolation (Figure 1).
Second, residues of theC-NOI that are critical for interactionwith
AvrB (Desveaux et al., 2007) and function of RPM1 (Chung et al.,
2011; Liu et al., 2011), as well as comparable residues in the
N-NOI, are surface-exposed (see Supplemental Figures 13B and
13C online). Third, the C-terminal cysteines required for acylation
and membrane targeting of RIN4 (Kim et al., 2005a; Takemoto
and Jones, 2005) and the AvrRpt2 cleavage sites in N-NOI and
C-NOI (Chisholm et al., 2005) are all exposed (see Supplemental
Figures 13B, 13D, and 13E online). An interesting feature of the
model is that the two NOIs interact extensively with one another
(see Supplemental Figure 13B online). Physical contact between
NOIs could contribute to the observed intermolecular interac-
tions between RIN4 homologs of soybeans (Selote and Kachroo,
2010).We hypothesized that the inability of 1D31 to suppress PTI
resulted frommisfolding. To test this idea, we obtained structural
predictions of 1D31 and 1D64 from I-TASSER. Indeed, 1D31 is
predicted to be mostly unstructured, whereas significant struc-
ture is restored in 1D64 (see Supplemental Figure 16 online).
1D64 retains some secondary structure elements that are pres-
ent in RIN4FL and are absent in 1D31, including an a helix
containing Gly127-Lys128. Although 1D31 is predicted to be
predominantly unstructured, the C terminus including the C-NOI
domain is predicted to resemble that of RIN4FL at the secondary
structure level (see Supplemental Figure 16C online). Hence, the
in vivo and modeling results are consistent with the partially
structured C terminus of 1D31 being sufficient to support RPM1
function, whereas the lack of broader structure precludes PTI
suppression (see Supplemental Figures 5 and 6 online).
3806 The Plant Cell
Our work provides new ideas about the targeting of RIN4 by
T3Es. We have recently shown that AvrRpm1 and AvrB induce
phosphorylation of RIN4 T166, and phosphorylation of this residue
elicits RPM1 activation (Chung et al., 2011). T3E-induced phos-
phorylationof T166 is performed, at least in part, byRIPK (Liu et al.,
2011). NOI domains have a conserved Thr or Ser at the position
comparable with T166 in RIN4. The hypothesis that the activity of
NOI proteins is controlled, at least in part, via phosphorylation of
this residue is supported by the role of T166 phosphorylation in
control of RPM1activation (Chung et al., 2011; Liu et al., 2011).We
hypothesize that phosphorylation of RIN4 on T166will enhance its
defense-suppressing activity. Also of interest is how AvrRpt2
might enhance the defense-suppressing activity of RIN4. AvrRpt2
is a protease that cleaves RIN4 at two locations. It seemed
counterintuitive that AvrRpt2 cleaves a negative regulator of PTI.
However, rather than simply inactivating or eliminating RIN4, our
data support a model in which cleavage by AvrRpt2 produces
active fragments of RIN4. Indeed, the twomain cleavage products
produced by AvrRpt2, ACP2 and ACP3, can be detected after
cleavage of native RIN4 by AvrRpt2 and are potent defense
suppressorswhen inducibly expressed. These findings, alongwith
the prior observations that native RIN4 negatively regulates PTI in
the absence of any T3E-mediated perturbation (Kim et al., 2005b)
and that RIN4 is a required target of the virulence activity of HopF2
(Wilton et al., 2010), support the hypothesis thatRIN4 isabonafide
virulence target of T3Es rather than a decoy (van der Hoorn and
Kamoun, 2008). Similar to RPM1 being activated by AvrRpm1- or
AvrB-induced phosphorylation of RIN4, RPS2 may respond to
AvrRpt2-induced cleavage product(s) of RIN4. In particular, cleav-
age-induced release of ACP2 from the membrane may be a key
step in activation of RPS2. Thus, as predicted by the “guard
hypothesis,” T3E-inducedperturbationsofRIN4 that suppressPTI
may also serve as elicitors of ETI in plants expressing RPM1 or
RPS2.
METHODS
Plants and Growth Conditions
Arabidopsis thaliana Col-0 wild-type plants were used in this study. The
mutants generated from Col-0 plants were as follows: rps2-101C has a
stop codon at amino acid 235 of RPS2 (Bent et al., 1994); rpm1-3 has a
stop codon at amino acid 87 of RPM1 (Grant et al., 1995); rin4 has
a T-DNA insertion after amino acid 146 of RIN4 (Mackey et al., 2003); the
rpm1 rps2 rin4 triple mutant was identified by marker-assisted breeding
(Belkhadir et al., 2004). Plants were grown on Metro Mix 360 (Sun Gro
Horticulture) at 258C/168C under an 8-h-light/16-h-dark cycle.
DNAManipulation and Generation of Transgenic Plants
All T7-tagged RIN4 derivatives were produced by overlap extension PCR
with full length RIN4 cDNA as the template (Aiyar et al., 1996). Overlap
extension PCRproductswere digestedwithNcoI (in the extremeN terminus
overlappingATG in the T7-tag) and XbaI (at the extremeC terminus after the
stop codon) and cloned downstream from the RIN4 native promoter in
pMAC100c (a PUC19-based vector containing 1340 bp of RIN4 promoter
from Col-0 up to the naturally occurring NcoI site at the RIN4 ATG).
To generate plants inducibly expressing RIN4 derivatives, the encoding
fragments (without the RIN4 native promoter) were subcloned from
pMAC100cvectors into theDex-induciblebinary vector, PTA7002 (Aoyama
and Chua, 1997). The resulting plasmids were introduced into Agrobacte-
rium tumefaciens strainGV3101byelectroporation. Transgenic plantswere
generated by vacuum-assisted floral dipping (Clough and Bent, 1998) of
flowering Col-0 or rpm1 rps2 rin4 plants into GV3101 carrying individual
plasmids. Transgenic progeny were selected by growth on Gamborg’s B5
(Invitrogen) agar plates containing 20 mM hygromycin B (Sigma-Aldrich).
Independent lines with single insertion loci, as determined by 3:1 segrega-
tion of hygromycin B resistance in the T2 generation, were selected and
propagated to homozygosity. Expression of FL:RIN4 and RIN4 derivatives
was induced by spraying plants with 20 mM Dex (Sigma-Aldrich) plus
0.005% Silwet L-77 solution (Lehle Seeds).
Bacterial Growth Assays
The hrcC T3S-deficient mutant of PstDC3000 (formerly hrpH) was used in
this study (Yuan and He, 1996). A suspension of 105 CFU/mL of hrcC in 10
mM MgCl2 was pressure-infiltrated with a needleless syringe into the
leaves of 4- to 5-week-old plants. After the infiltration, the leaves were
allowed to dry (;4 h), and the plants were kept in 100% humidity (by
covering with a clear dome) for the remainder of the experiment. For each
measurement, nine leaf discs were separated into three samples (three
discs per tube), and the titers from these three samples were used to
perform statistical analysis. Leaf discs were collected from the infiltrated
area, ground in 10 mM MgCl2, and serially diluted to measure bacterial
numbers. The RIN4 transgenes were induced by spraying 20 mM Dex
(Sigma-Aldrich) solution containing 0.005% (v/v) Silwet L-77 (Lehle) 48 h
before bacterial infiltration. P values were calculated using Student’s t
test. All growth assays are representative of at least three independent
biological replicates.
Protein
Total protein extraction and cell fractionations were performed as de-
scribed previously (Mackey et al., 2002). Briefly, total protein extracts were
prepared by grinding;2 cm2 of tissue per 0.2mLof grindingbuffer (20mM
Tris-HCl [pH 7.5]), 150mMNaCl, 1mMEDTA, 1%Triton X-100, 0.1%SDS,
5 mM DTT, and plant protease inhibitor cocktail (Sigma-Aldrich) and
pelleting insoluble debris by centrifugation at 20,000 3 g for 10 min at
48C. Concentration of protein in the supernatant was determined by using
the Bio-Rad protein assay reagent (Bio-Rad). Samples, typically 30 mg,
were separated onSDS-PAGEgels (mini protean; Bio-Rad) and transferred
to polyvinylidene fluoride membrane. All SDS-PAGE gels were 12 to 15%
acrylamide except in Supplemental Figure 12 online (10%). Immunoblots
weredonebystandardmethods. Anti-RIN4sera (Mackey et al., 2002), anti-
T7monoclonal antibody (Novagen), and anti–PR-1 sera (Kliebenstein et al.,
1999) were used at dilutions of 1:5000, 1:10,000, and 1:10,000, respec-
tively. Chemiluminescent detection and band quantification were done
using the ChemiDoc XRS system (Bio-Rad).
Subcellular Fractionation
Membrane proteins were fractionated by grinding 0.1 g of tissue per 1mL
of buffer (10 mM Tris-HCl [pH 7.0], 0.33 M Suc, 1 mM EDTA, and plant
protease inhibitor cocktail [Sigma-Aldrich]) and pelleting insoluble debris
by centrifugation at 20,0003 g for 20 min at 48C. The supernatant of this
spin constituted the total (T) fraction. A total of 10 mL of 1 M CaCl2 was
added to 500 mL of the total fraction, and the microsomal fraction was
pelleted at 25,000 3 g for 90 min at 48C. The supernatant from this spin
was the soluble (S) fraction. The pellet was resuspended in 30 mL of 13
SDS-PAGE loading dye, heated at 658C for 15 min. This constituted the
membrane (M) fraction.
Suppression of PTI by RIN4 3807
Callose Staining and Quantification
Leaves from 4-week-old plants were syringe-infiltrated with 30 mM flg22 or
distilled water andwere collected after 16 h. Transgenic lines were induced
by spraying 20 mM Dex containing 0.005% Silwet L-77 at 48 h before the
infiltration with flg22. Five leaves for each treatment were then stained with
aniline blue (Sigma-Aldrich) (Kim and Mackey, 2008). Whole leaves were
cleared by submersion in lactophenol alcohol (a mixture of 1 volume of a
1:1:1:1 volume mix of glycerol, saturated phenol, lactophenol, and deion-
ized water and 2 volumes of 95% ethanol) and incubated for 5 min at 958C
followed by an overnight incubation in fresh lactophenol alcohol at room
temperature. The cleared leaves were rinsed in 50% ethanol and then in
water and were then stained with 0.01% aniline blue in 150 mMphosphate
buffer (pH 9.5). Stained leaves were mounted in 50% glycerol and were
visualized under epifluorescent illumination by a Nikon eclipse 80i micro-
scope. Four pictures of different areas were taken of each leaf (for 20
images per treatment), and callose deposits were counted using the
ImageJ software (http://rsbweb.nih.gov/ij/).
Trypan Blue Staining
Trypan blue staining was performed as described previously (Koch and
Slusarenko, 1990). Briefly, leaves were submerged in a staining solution
composed of 1 part staining mix (1:1:1:1 mix of phenol, lactic acid,
glycerin, and water plus 0.05% [w/v] trypan blue) and 2 parts ethanol. The
leaves were incubated in the staining solution at 958C for 3 min followed
by an additional overnight incubation. Stained leaves were cleared in
15 M chloral hydrate solution and mounted in 70% glycerol.
DNAManipulation and Generation of Transgenic Plants
The evolutionary history was inferred using the Neighbor-Joining method
(Saitou andNei, 1987). The tree is drawn to scale with branch lengths in the
same units as those of the evolutionary distances used to infer the
phylogenetic tree. The evolutionary distances were computed using the
Dayhoff matrix based method (Schwartz and Dayhoff, 1979), and branch
lengths correspond to the number of amino acid substitutions per site. All
positions containing gaps and missing data were eliminated from the data
set (completedeletion option) so that therewere a total of 28positions in the
final data set. All phylogenetic analyseswereconducted inMEGA4 (Tamura
et al., 2007).
To generate plants expressing RIN4 derivatives under control of
the native promoter, the encoding fragments (with the 1340 bp RIN4 native
promoter) were subcloned from pMAC100c vectors into pBAR1 (promo-
terless binary vector conferring Basta resistance) and moved into GV3101
by electroporation. Transgenic plants were generated by vacuum-assisted
floral dipping (Clough and Bent, 1998) of flowering rpm1 rps2 rin4 RPM1-
myc plants (Boyes et al., 1998; Belkhadir et al., 2004). Transgenic progeny
were selected by spraying soil-grown seedlings on 3 consecutive days
with 0.04% (w/v) Basta and 0.005% Silwet L-77 solution. Independent T1
lines with single insertion loci, as determined by 3:1 segregation of Basta
resistance in the T2 generation, were selected and propagated to homo-
zygosity.
Bacteria, HR, and Conductance Measurements for Assessment of
RPM1 Function
PstDC3000 carried either pVSP61 or derivatives of this plasmid contain-
ing avrRpm1 or avrB. For HR and conductance assays, leaves were
infiltrated with a suspension of 53 107 CFU/mL bacteria in 10mMMgCl2.
For measurements of conductance, eight leaf discs (8 mm diameter)
were removed immediately after infiltration (time 0) and were floated in
25 mL of water to wash away ions released during leaf disc removal.
After 10 min, the wash water was replaced with 10 mL of fresh water,
and the conductance in microsiemens per centimeter was measured
over time.
ROS Accumulation
Four-week-old rpm1 rps2 rin4 plants expressing RIN4 derivatives and
control plants were sprayed with Dex (20 mM). After 36 h, eight leaf disks
(6.5 mm diameter) were excised from each plant and were floated on
distilled water for 4 to 6 h. Then three leaf disks were transferred to a
microfuge tube containing 100 mL of luminol solution, Immun-Star HRP
Substrate (Bio-Rad), and 1 mL of horseradish peroxidase-streptavidin
(Jackson Immunoresearch) and were supplemented with either 1 mL of
1 mM flg22 peptide (10 mM final concentration) or 1 mL water. Lumines-
cence was measured every 10 s for 18 min by a Glomax 20/20
luminometer (Promega). Within each biological replicate, the data were
normalized, with the average peak value from rpm1 rps2 rin4 plants
exposed to flg22 set to 1.
Real-Time PCR
Four- to five-week-old plants expressing RIN4 derivatives and control
plants were sprayed with Dex (20 mM). After 48 h, leaves were infiltrated
with flg22 (30 mM) or water. Samples were collected 2 h after infiltration
and were flash-frozen in liquid nitrogen. Total RNA was isolated using the
RNeasy PlantMini Kit (Qiagen) followed byDNase I (Invitrogen) treatment.
The RNA quality was determined by gel electrophoresis and quantitated
using a nano-dropmodel ND-1000 (ThermoScientific). First-strand cDNA
was synthesized from 1 mg of RNA using oligo-dT primer and AMV
reverse transcriptase (Promega). Quantitative real-time PCR analyses
were performed using aBio-Rad iQ5 real-timePCRdetection systemwith
iQ SYBR green supermix (Bio-Rad). Actin was used as a control, and data
were analyzed using iQ5 software (Bio-Rad). Primers used for real-time
PCR are listed in Supplemental Table 1 online.
Computational Modeling
The computation models for RIN4FL and the RIN4 derivatives were built
by submitting primary sequences to the I-TASSER server (http://zhang.
bioinformatics.ku.edu/I-TASSER). The I-TASSER server was ranked
number 1 during the Critical Assessment of Techniques for Protein
Structure Prediction experiment three assessments in a row (CASP 2006,
CASP 2008, and CASP 2010). The model was generated using a meta-
threading approach followed by Monte Carlo simulations, with unaligned
regions built by ab initio modeling (Zhang, 2008). To generate the RIN4FL
model, the following six proteins were used as templates: 1m11_R
(human decay-accelerating factor/coat protein), 2nud (AvrB complexed
with a high-affinity RIN4 peptide), 1nkw_B (large ribosomal subunit from
Deinococcus radiodurans), 2dpm_A (DpnM DNA adenine methyltrans-
ferase from the DpnII restriction system of Streptococcus pneumoniae
bound to S-adenosylmethionine), 1mv3_A (Myc box–dependent inter-
acting protein 1), and 2qzu_A (putative sulfatase yidJ from Bacteroides
fragilis). Visualization and optimization of the predicted model was done
using the Jmol server (http://firstglance.jmol.org) and Chime (http://www.
symyx.com/downloads). The PROCHECK software suite (http://www.
ebi.ac.uk/thornton-srv/software/PROCHECK/) was used to generate the
Ramachandran plot and to assign residues to the favored, allowed, and
disallowed regions.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
3808 The Plant Cell
numbers: RIN4 homologs from other plant species (and accession num-
bers) are Arabidopsis lyrata (ABR46111.1), Brassica juncea (ABM30198),
Medicago truncatula (ACJ83941), Lactuca saligna (GQ497776), Solanum
lycopersicum (TC174419), Populus trichocarpa (XM_002316554), Vitis vi-
nifera (CBI35706), Oryza sativa (NM_001058429), Zea mays
(NP_001152021), Brachypodium distachyon (Bradi3g40950), and Physco-
mitrella patens (XP_001766424). Arabidopsis genes analyzed in this study
are: RIN4 (AT3G25070), MYB51 (AT1G18570), FRK1 (AT2G19190), ACT2
(AT3G18780).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Accumulation of ACP2 and ACP3 upon
Cleavage of Native RIN4 by AvrRpt2.
Supplemental Figure 2. Residues between the NOI Domains Are Not
Essential for PTI Suppression by RIN4.
Supplemental Figure 3. Membrane Association of RIN4 Derivatives
Depends on the C-Terminal Cysteines.
Supplemental Figure 4. Alignment of NOI Domains.
Supplemental Figure 5. A Small N-Terminal Deletion Disrupts the
PTI-Suppressing Activity of RIN4.
Supplemental Figure 6. 1D31 Retains the Ability to Support RPM1
Function.
Supplemental Figure 7. Alignment of the C Termini of RIN4 Homo-
logs and NOI-Containing Proteins.
Supplemental Figure 8. Expression of RIN4 Derivatives in Plants
Screened for Use in Figure 5A.
Supplemental Figure 9. Expression of RIN4 Derivatives in Plants
Screened for Use in Figure 5B and Supplemental Figure 10.
Supplemental Figure 10. Flg22-Induced ROS Accumulation in Plants
Expressing RIN4 Derivatives.
Supplemental Figure 11. Flg22-Induced Expression of FRK1 and
MYB51 in Plants Expressing RIN4 Derivatives.
Supplemental Figure 12. Summary of Results Obtained from RIN4
Structure:Function Analysis.
Supplemental Figure 13. Structural Model of RIN4.
Supplemental Figure 14. RIN4 Is Modeled as Globular in Additional
Structural Predictions.
Supplemental Figure 15. Validation of Structural Model of RIN4FL by
Ramachandran Plot.
Supplemental Figure 16. Ribbon Diagrams of Predicted Structures
of RIN4FL, 1D31, and 1D64.
Supplemental Table 1. List of Primers Used for Real-Time PCR.
Supplemental Data Set 1. Text File of Alignment Used to Generate
Tree in Figure 3.
ACKNOWLEDGMENTS
This work was supported by funding to D.M. from the National Science
Foundation (MCB-0718882) and the Ohio Agricultural Research and
Development Center. L.D.C. was supported by an Excellence in PMBB
Fellowship from the Plant Molecular Biology and Biotechnology Pro-
gram at Ohio State University.
AUTHOR CONTRIBUTIONS
A.J.A., L.d.C., and D.M. designed the research. A.J.A. and L.d.C. performed
research. A.J.A., L.d.C., and D.M. analyzed data and wrote the article.
Received June 29, 2011; revised August 26, 2011; accepted September
15, 2011; published October 7, 2011.
REFERENCES
Aiyar, A., Xiang, Y., and Leis, J. (1996). Site-directed mutagenesis
using overlap extension PCR. Methods Mol. Biol. 57: 177–191.
Alfano, J.R., and Collmer, A. (2004). Type III secretion system effector
proteins: Double agents in bacterial disease and plant defense. Annu.
Rev. Phytopathol. 42: 385–414.
Aoyama, T., and Chua, N.-H. (1997). A glucocorticoid-mediated tran-
scriptional induction system in transgenic plants. Plant J. 11: 605–612.
Axtell, M.J., and Staskawicz, B.J. (2003). Initiation of RPS2-specified
disease resistance in Arabidopsis is coupled to the AvrRpt2-directed
elimination of RIN4. Cell 112: 369–377.
Axtell, M.J., Chisholm, S.T., Dahlbeck, D., and Staskawicz, B.J.
(2003). Genetic and molecular evidence that the Pseudomonas
syringae type III effector protein AvrRpt2 is a cysteine protease.
Mol. Microbiol. 49: 1537–1546.
Belkhadir, Y., Nimchuk, Z., Hubert, D.A., Mackey, D., and Dangl, J.L.
(2004). Arabidopsis RIN4 negatively regulates disease resistance
mediated by RPS2 and RPM1 downstream or independent of the
NDR1 signal modulator and is not required for the virulence functions
of bacterial type III effectors AvrRpt2 or AvrRpm1. Plant Cell 16: 2822–
2835.
Bent, A.F., Kunkel, B.N., Dahlbeck, D., Brown, K.L., Schmidt, R.,
Giraudat, J., Leung, J., and Staskawicz, B.J. (1994). RPS2 of
Arabidopsis thaliana: a leucine-rich repeat class of plant disease
resistance genes. Science 265: 1856–1860.
Boyes, D.C., Nam, J., and Dangl, J.L. (1998). The Arabidopsis thaliana
RPM1 disease resistance gene product is a peripheral plasma mem-
brane protein that is degraded coincident with the hypersensitive
response. Proc. Natl. Acad. Sci. USA 95: 15849–15854.
Chinchilla, D., Boller, T., and Robatzek, S. (2007). Flagellin signalling
in plant immunity. Adv. Exp. Med. Biol. 598: 358–371.
Chisholm, S.T., Coaker, G., Day, B., and Staskawicz, B.J. (2006).
Host-microbe interactions: Shaping the evolution of the plant immune
response. Cell 124: 803–814.
Chisholm, S.T., Dahlbeck, D., Krishnamurthy, N., Day, B., Sjolander,
K., and Staskawicz, B.J. (2005). Molecular characterization of pro-
teolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2.
Proc. Natl. Acad. Sci. USA 102: 2087–2092.
Chung, E.H., da Cunha, L., Wu, A.J., Gao, Z., Cherkis, K., Afzal, A.
J., Mackey, D., and Dangl, J.L. (2011). Specific threonine
phosphorylation of a host target by two unrelated type III effectors
activates a host innate immune receptor in plants. Cell Host Microbe
9: 125–136.
Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
J. 16: 735–743.
Cui, H., Wang, Y., Xue, L., Chu, J., Yan, C., Fu, J., Chen, M., Innes,
R.W., and Zhou, J.M. (2010). Pseudomonas syringae effector protein
AvrB perturbs Arabidopsis hormone signaling by activating MAP
kinase 4. Cell Host Microbe 7: 164–175.
da Cunha, L., Sreerekha, M.V., and Mackey, D. (2007). Defense
suppression by virulence effectors of bacterial phytopathogens. Curr.
Opin. Plant Biol. 10: 349–357.
Suppression of PTI by RIN4 3809
Day, B., Dahlbeck, D., Huang, J., Chisholm, S.T., Li, D., and
Staskawicz, B.J. (2005). Molecular basis for the RIN4 negative
regulation of RPS2 disease resistance. Plant Cell 17: 1292–1305.
Desveaux, D., Singer, A.U., Wu, A.J., McNulty, B.C., Musselwhite, L.,
Nimchuk, Z., Sondek, J., and Dangl, J.L. (2007). Type III effector
activation via nucleotide binding, phosphorylation, and host target
interaction. PLoS Pathog. 3: e48.
Felix, G., Duran, J.D., Volko, S., and Boller, T. (1999). Plants have a
sensitive perception system for the most conserved domain of bac-
terial flagellin. Plant J. 18: 265–276.
Geng, X., and Mackey, D. (2011). Dose–response to and systemic
movement of dexamethasone in the GVG-inducible transgene system
in Arabidopsis. Methods Mol. Biol. 712: 59–68.
Grant, M.R., Godiard, L., Straube, E., Ashfield, T., Lewald, J., Sattler,
A., Innes, R.W., and Dangl, J.L. (1995). Structure of the Arabidopsis
RPM1 gene enabling dual specificity disease resistance. Science 269:
843–846.
Guo, M., Tian, F., Wamboldt, Y., and Alfano, J.R. (2009). The majority
of the type III effector inventory of Pseudomonas syringae pv. tomato
DC3000 can suppress plant immunity. Mol. Plant Microbe Interact.
22: 1069–1080.
Hauck, P., Thilmony, R., and He, S.Y. (2003). A Pseudomonas
syringae type III effector suppresses cell wall-based extracellular
defense in susceptible Arabidopsis plants. Proc. Natl. Acad. Sci.
USA 100: 8577–8582.
Jones, J.D., and Dangl, J.L. (2006). The plant immune system. Nature
444: 323–329.
Kim, H.S., Desveaux, D., Singer, A.U., Patel, P., Sondek, J., and
Dangl, J.L. (2005a). The Pseudomonas syringae effector AvrRpt2
cleaves its C-terminally acylated target, RIN4, from Arabidopsis mem-
branes to block RPM1 activation. Proc. Natl. Acad. Sci. USA 102:
6496–6501.
Kim, M.G., and Mackey, D. (2008). Measuring cell-wall-based defenses
and their effect on bacterial growth in Arabidopsis. Methods Mol. Biol.
415: 443–452.
Kim, M.G., Geng, X., Lee, S.Y., and Mackey, D. (2009). The Pseudo-
monas syringae type III effector AvrRpm1 induces significant defenses
by activating the Arabidopsis nucleotide-binding leucine-rich repeat
protein RPS2. Plant J. 57: 645–653.
Kim, M.G., da Cunha, L., McFall, A.J., Belkhadir, Y., DebRoy, S.,
Dangl, J.L., and Mackey, D. (2005b). Two Pseudomonas syringae
type III effectors inhibit RIN4-regulated basal defense in Arabidopsis.
Cell 121: 749–759.
Kliebenstein, D.J., Dietrich, R.A., Martin, A.C., Last, R.L., and Dangl,
J.L. (1999). LSD1 regulates salicylic acid induction of copper zinc
superoxide dismutase in Arabidopsis thaliana. Mol. Plant Microbe
Interact. 12: 1022–1026.
Koch, E., and Slusarenko, A. (1990). Arabidopsis is susceptible to
infection by a downy mildew fungus. Plant Cell 2: 437–445.
Lim, M.T., and Kunkel, B.N. (2004). Mutations in the Pseudomonas
syringae avrRpt2 gene that dissociate its virulence and avirulence
activities lead to decreased efficiency in AvrRpt2-induced disappear-
ance of RIN4. Mol. Plant Microbe Interact. 17: 313–321.
Liu, J., Elmore, J.M., Lin, Z.J., and Coaker, G. (2011). A receptor-like
cytoplasmic kinase phosphorylates the host target RIN4, leading to
the activation of a plant innate immune receptor. Cell Host Microbe 9:
137–146.
Liu, J., Elmore, J.M., Fuglsang, A.T., Palmgren, M.G., Staskawicz,
B.J., and Coaker, G. (2009). RIN4 functions with plasma membrane H
+-ATPases to regulate stomatal apertures during pathogen attack.
PLoS Biol. 7: e1000139.
Luo, Y., Caldwell, K.S., Wroblewski, T., Wright, M.E., and Michelmore,
R.W. (2009). Proteolysis of a negative regulator of innate immunity is
dependent on resistance genes in tomato and Nicotiana benthamiana
and induced by multiple bacterial effectors. Plant Cell 21: 2458–2472.
Mackey, D., and McFall, A.J. (2006). MAMPs and MIMPs: Proposed
classifications for inducers of innate immunity. Mol. Microbiol. 61:
1365–1371.
Mackey, D., Holt III, B.F., Wiig, A., and Dangl, J.L. (2002). RIN4 interacts
with Pseudomonas syringae type III effector molecules and is required
for RPM1-mediated resistance in Arabidopsis. Cell 108: 743–754.
Mackey, D., Belkhadir, Y., Alonso, J.M., Ecker, J.R., and Dangl, J.L.
(2003). Arabidopsis RIN4 is a target of the type III virulence effector
AvrRpt2 and modulates RPS2-mediated resistance. Cell 112: 379–
389.
Maleck, K., Levine, A., Eulgem, T., Morgan, A., Schmid, J., Lawton,
K.A., Dangl, J.L., and Dietrich, R.A. (2000). The transcriptome of
Arabidopsis thaliana during systemic acquired resistance. Nat. Genet.
26: 403–410.
McHale, L., Tan, X., Koehl, P., and Michelmore, R.W. (2006). Plant
NBS-LRR proteins: Adaptable guards. Genome Biol. 7: 212.
Mersmann, S., Bourdais, G., Rietz, S., and Robatzek, S. (2010).
Ethylene signaling regulates accumulation of the FLS2 receptor and is
required for the oxidative burst contributing to plant immunity. Plant
Physiol. 154: 391–400.
Morris, A.L., MacArthur, M.W., Hutchinson, E.G., and Thornton, J.M.
(1992). Stereochemical quality of protein structure coordinates. Pro-
teins 12: 345–364.
Saitou, N., and Nei, M. (1987). The neighbor-joining method: A newmethod
for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425.
Schwartz, R.M., and Dayhoff, M.O. (1979). Protein and nucleic acid
sequence data and phylogeny. Science 205: 1038–1039.
Selote, D., and Kachroo, A. (2010). RPG1-B-derived resistance to
AvrB-expressing Pseudomonas syringae requires RIN4-like proteins
in soybean. Plant Physiol. 153: 1199–1211.
Shang, Y., Li, X., Cui, H., He, P., Thilmony, R., Chintamanani, S.,
Zwiesler-Vollick, J., Gopalan, S., Tang, X., and Zhou, J.M. (2006).
RAR1, a central player in plant immunity, is targeted by Pseudomo-
nas syringae effector AvrB. Proc. Natl. Acad. Sci. USA 103: 19200–
19205.
Shen, Q.H., Saijo, Y., Mauch, S., Biskup, C., Bieri, S., Keller, B., Seki,
H., Ulker, B., Somssich, I.E., and Schulze-Lefert, P. (2007). Nuclear
activity of MLA immune receptors links isolate-specific and basal
disease-resistance responses. Science 315: 1098–1103.
Takemoto, D., and Jones, D.A. (2005). Membrane release and desta-
bilization of Arabidopsis RIN4 following cleavage by Pseudomonas
syringae AvrRpt2. Mol. Plant Microbe Interact. 18: 1258–1268.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4:
Molecular Evolutionary Genetics Analysis (MEGA) software version
4.0. Mol. Biol. Evol. 24: 1596–1599.
Tao, Y., Xie, Z., Chen, W., Glazebrook, J., Chang, H.S., Han, B., Zhu,
T., Zou, G., and Katagiri, F. (2003). Quantitative nature of Arabi-
dopsis responses during compatible and incompatible interactions
with the bacterial pathogen Pseudomonas syringae. Plant Cell 15:
317–330.
Tsuda, K., Sato, M., Glazebrook, J., Cohen, J.D., and Katagiri, F.
(2008). Interplay between MAMP-triggered and SA-mediated defense
responses. Plant J. 53: 763–775.
van der Hoorn, R.A., and Kamoun, S. (2008). From Guard to decoy: A
new model for perception of plant pathogen effectors. Plant Cell 20:
2009–2017.
Wan, J., Zhang, X.C., Neece, D., Ramonell, K.M., Clough, S., Kim,
S.Y., Stacey, M.G., and Stacey, G. (2008). A LysM receptor-like
kinase plays a critical role in chitin signaling and fungal resistance in
Arabidopsis. Plant Cell 20: 471–481.
Wang, Y., Li, J., Hou, S., Wang, X., Li, Y., Ren, D., Chen, S., Tang, X.,
3810 The Plant Cell
and Zhou, J.M. (2010). A Pseudomonas syringae ADP-ribosyltrans-
ferase inhibits Arabidopsis mitogen-activated protein kinase kinases.
Plant Cell 22: 2033–2044.
Wilton, M., Subramaniam, R., Elmore, J., Felsensteiner, C., Coaker,
G., and Desveaux, D. (2010). The type III effector HopF2Pto targets
Arabidopsis RIN4 protein to promote Pseudomonas syringae viru-
lence. Proc. Natl. Acad. Sci. USA 107: 2349–2354.
Yuan, J., and He, S.Y. (1996). The Pseudomonas syringae Hrp regu-
lation and secretion system controls the production and secretion of
multiple extracellular proteins. J. Bacteriol. 178: 6399–6402.
Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction.
BMC Bioinformatics 9: 40.
Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller,
T., and Felix, G. (2006). Perception of the bacterial PAMP EF-Tu by
the receptor EFR restricts Agrobacterium-mediated transformation.
Cell 125: 749–760.
Suppression of PTI by RIN4 3811
DOI 10.1105/tpc.111.088708; originally published online October 7, 2011; 2011;23;3798-3811Plant Cell
Ahmed J. Afzal, Luis da Cunha and David MackeyPAMP-Triggered Immunity
RIN4 Regulate Its Suppression ofArabidopsisSeparable Fragments and Membrane Tethering of
This information is current as of October 4, 2020
Supplemental Data /content/suppl/2011/09/30/tpc.111.088708.DC1.html
References /content/23/10/3798.full.html#ref-list-1
This article cites 56 articles, 21 of which can be accessed free at:
Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs http://www.plantcell.org/cgi/alerts/ctmain
Sign up for eTOCs at:
CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain
Sign up for CiteTrack Alerts at:
Subscription Information http://www.aspb.org/publications/subscriptions.cfm
is available at:Plant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists